Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

Pin1 and secondary hyperparathyroidism of chronic kidney disease: gene polymorphisms and protein levels.

BACKGROUND: Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a key regulator of PTH mRNA stability. Secondary hyperparathyroidism (SHPT), which is characterized by elevated serum PTH levels, is a common complication of CKD. We investigated the possible associations between CKD with SHPT (CKD SHPT) and single-nucleotide polymorphisms of the Pin1 gene and compared the levels of the Pin1 protein in the CKD SHPT patients with those of the controls. METHODS: The study group included 251 CKD SHPT patients and 61 controls. One putative functional SNP (single nucleotide polymorphism) in the Pin1 promoter (rs2233679C > T: c.-667C > T) is the main object. Genotyping was performed on purified DNA using polymerase chain reaction-restriction (PCR) and restriction fragment length polymorphisms (RFLP). The levels of Pin1 were measured in serum using an enzyme-linked immunosorbent assay. RESULTS: Genotyping showed that CT + TT in the Pin1 promoter was significantly more common in the CKD SHPT group than in the control group (p<.05). The correlation analysis demonstrated that a significant difference in the C to T transition in the Pin1 promoter contributed to CKD SHPT (χ2=12.47, p<.05; Odds ratios (OR) = 1.26, 95% confidence (CI) intervals =1.06-1.49). The multivariate logistic regression analysis reported that the OR and 95%CI were 12.693 and 2.029-75.819 (p<.05), respectively, in the Pin1 gene promoter -667T variant genotypes (CT + TT) after adjusting for other factors, and those values in Pin1 were 0.310 and 0.122-0.792 (p<.05). CONCLUSION: The -667T genetic variants in the Pin1 promoter contribute to an increased risk of CKD SHPT and may be biomarkers of susceptibility to CKD SHPT.

Discovery of a potent and selective ROMK inhibitor with improved pharmacokinetic properties based on an octahydropyrazino[2,1-c][1,4]oxazine scaffold.

Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the discovery of analogs with improved pharmacokinetic profiles. This new series also retained comparable ROMK potency compared to earlier leads.

Accelerated Age-Related Arterial Stiffness in Systemic Lupus Erythematosus Patients.

BACKGROUND: An increased prevalence of arterial stiffness in systemic lupus erythematosus (SLE) patients has been established, but the mechanisms of progression of arterial stiffness with increasing age have not been fully explored. OBJECTIVES: To investigate age-related progression of arterial stiffness among SLE patients relative to healthy controls. METHODS: A total of 161 female SLE patients who were enrolled in the Chinese SLE Treatment and Research group (CSTAR) registry and 135 age-matched healthy control subjects participated in this cross-sectional investigation. Traditional cardiovascular risk factors and SLE-related parameters were assessed on the day that brachial-ankle pulse wave velocity (baPWV) was examined. SAS 9.3 was used to perform all statistical analyses in this study. Linear regression and curvilinear regression models were utilized to analyze the association between age and arterial stiffness. RESULTS: Arterial stiffness based on baPWV significantly differed between the SLE patients and controls in the different age groups, and within the SLE group. The baPWV increments for each age group (<25, 25-34, 35-45, and >45) were 30 cm/s, 52 cm/s, and 121 cm/s for the controls and 61 cm/s, 132 cm/s, and 155 cm/s for the SLE patients, respectively. Curvilinear regression and linear regression revealed various trends of increased arterial stiffness among the SLE patients compared with the healthy controls. The correlation coefficients between age and arterial stiffness significantly differed among the SLE patients relative to the healthy controls (correlation coefficients of 0.46478 and 0.52612, respectively; t = 2.05; P = 0.0409). Multivariate analysis revealed that age, mean blood pressure (BP) (P < 0.0001), erythrocyte sedimentation rate (ESR) (P = 0.0073), prednisone course (P = 0.0144), and SLE disease activity (P = 0.0405) were associated with arterial stiffness among the SLE patients. Further, these patients exhibited earlier exposure to and higher frequencies of several risk factors compared with the controls in each age group (<25 years: OR = 6.3253; 25-34 years: OR = 3.1043; 35-45 years: OR = 3.1316; >45 years: OR = 3.6020). CONCLUSIONS: The mechanisms of the age-related progression of arterial stiffness differed among the SLE patients relative to the healthy controls. Furthermore, accelerated arterial stiffness was observed among the SLE patients relative to the healthy controls with advancing age.

The importin α subunit PsIMPA1 mediates the oxidative stress response and is required for the pathogenicity of Phytophthora sojae.

The sensing of extracellular signals and their transduction into an appropriate response are crucial for the survival and virulence of plant pathogens. Eukaryotic plant pathogens must overcome the obstacles posed by nuclear membranes to manipulate gene expression to adapt to the host challenge. A highly sophisticated mechanism is the use of importins to transport proteins into the nucleus. In this study, we identified a conserved importin α gene, PsIMPA1, in Phytophthora sojae that was differentially expressed during the life cycle of this soybean pathogen. PsIMPA1 expression was lowest in zoospores and cysts but relatively consistent during the other life cycle stages, except for a slight increase at 6h post infection. Silenced mutants Psimpa1 had a decreased growth rate, an aberrant mycelial morphology, and a severely impaired ability to form oospores and sporangia. In addition, the Psimpa1 mutants exhibited reduced pathogenicity compared to the wild type. 3,3-Diaminobenzidine (DAB) staining and in vitro hydrogen peroxide tolerance assays showed that the scavenging of reactive oxygen species by these mutants was significantly impaired. Taken together, these results indicate that PsIMPA1 regulates multiple processes during the life cycle of P. sojae.

Guided reconstitution of membrane protein fragments.

Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs. Short peptides representing the N-terminal (NT) and C-terminal (CT) regions of the first extracellular loop (EL1) of Ste2p, the Saccharomyces cerevisiae alpha-factor mating receptor, were activated using these reagents and the efficiencies of activation and rates of heterodimerization were analyzed. Activation of NT peptides with DTNP and 4-PDS resulted in about 60% yield, but heterodimerization was rapid and nearly quantitative. Double transmembrane domain protein fragments were biosynthesized and used in Guided Reconstitution reactions. A 102-residue fragment, 2TM-tail [Ste2p(G31-I120C)], was heterodimerized with CT-EL1-tail(DTNP) at pH 4.6 with a yield of ∼75%. A 132-residue fragment, 2TMlong-tail [Ste2p(M1-I120C)], was expressed in both unlabeled and (15)N-labeled forms and used with a peptide comprising the third transmembrane domain, to generate a 180-residue segmentally labeled 3TM protein that was found to be segmentally labeled using [(15)N,(1)H]-HSQC analysis. Our data indicate that the Guided Reconstitution method would be applicable to the segmental labeling of a membrane protein with 3 transmembrane domains and may prove useful in the preparation of an intact reconstituted GPCR for use in biophysical analysis and structure determination.

3D-printed Bi2Te3-based Thermoelectric Generators for Energy Harvesting and Temperature Response.

Thermoelectric generators (TEGs) are environmentally friendly energy harvesting technologies that hold great promise in the field of self-powered electronics and sensing. However, the current development of thermoelectric (TE) devices has largely lagged behind the development of thermoelectric materials, especially in the preparation of thermoelectric components with customizable shapes and excellent properties, which largely limits their practical applications. These issues can be effectively addressed by using 3D printing technology. Here, we print multiple p-type thermoelectric legs (pins) consecutively with this simple technique, and the printed TEGs have excellent thermal potential (288 µV K-1 at room temperature) and excellent temperature response properties, which exhibited an output voltage of 127.94 mV at a temperature difference (ΔT) of 40 K. The 3D-printed thermoelectric generator enables the collection of thermal energy. In addition, the device has excellent temperature sensing characteristics, and this temperature signal to electrical signal conversion is very rapid, which enables temperature sensing alarms in a wide temperature domain. Combining these features, an energy harvesting and electrical alarm concept for home-scale applications is proposed, which is expected to provide a diverse research idea for the application of next-generation thermoelectric devices.

Structure Guided Discovery of Novel Pan Metallo-ß-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

IGTCM: An integrative genome database of traditional Chinese medicine plants.

Fully understanding traditional Chinese medicines (TCMs) is still challenging because of the extreme complexity of their chemical components and mechanisms of action. The TCM Plant Genome Project aimed to obtain genetic information, determine gene functions, discover regulatory networks of herbal species, and elucidate the molecular mechanisms involved in the disease prevention and treatment, thereby accelerating the modernization of TCMs. A comprehensive database that contains TCM-related information will provide a vital resource. Here, we present an integrative genome database of TCM plants (IGTCM) that contains 14,711,220 records of 83 annotated TCM-related herb genomes, including 3,610,350 genes, 3,534,314 proteins and corresponding coding sequences, and 4,032,242 RNAs, as well as 1033 non-redundant component records for 68 herbs, downloaded and integrated from the GenBank and RefSeq databases. For minimal interconnectivity, each gene, protein, and component was annotated using the eggNOG-mapper tool and Kyoto Encyclopedia of Genes and Genomes database to acquire pathway information and enzyme classifications. These features can be linked across several species and different components. The IGTCM database also provides visualization and sequence similarity search tools for data analyses. These annotated herb genome sequences in IGTCM database are a necessary resource for systematically exploring genes related to the biosynthesis of compounds that have significant medicinal activities and excellent agronomic traits that can be used to improve TCM-related varieties through molecular breeding. It also provides valuable data and tools for future research on drug discovery and the protection and rational use of TCM plant resources. The IGTCM database is freely available at http://yeyn.group:96/.

Discovery of benzodihydroisofurans as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase inhibitors.

A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure-activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.

Emerging Flexible Thermally Conductive Films: Mechanism, Fabrication, Application.

Effective thermal management is quite urgent for electronics owing to their ever-growing integration degree, operation frequency and power density, and the main strategy of thermal management is to remove excess energy from electronics to outside by thermal conductive materials. Compared to the conventional thermal management materials, flexible thermally conductive films with high in-plane thermal conductivity, as emerging candidates, have aroused greater interest in the last decade, which show great potential in thermal management applications of next-generation devices. However, a comprehensive review of flexible thermally conductive films is rarely reported. Thus, we review recent advances of both intrinsic polymer films and polymer-based composite films with ultrahigh in-plane thermal conductivity, with deep understandings of heat transfer mechanism, processing methods to enhance thermal conductivity, optimization strategies to reduce interface thermal resistance and their potential applications. Lastly, challenges and opportunities for the future development of flexible thermally conductive films are also discussed.

Identification of residue-to-residue contact between a peptide ligand and its G protein-coupled receptor using periodate-mediated dihydroxyphenylalanine cross-linking and mass spectrometry.

Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ∼65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.

Discovery of benzimidazole pyrrolidinyl amides as prolylcarboxypeptidase inhibitors.

A series of benzimidazole pyrrolidinyl amides containing a piperidinyl group were discovered as novel prolylcarboxypeptidase (PrCP) inhibitors. Low-nanomolar IC(50)'s were achieved for several analogs, of which compound 9b displayed modest ex vivo target engagement in eDIO mouse plasma. Compound 9b was also studied in vivo for its effect on weight loss and food intake in an eDIO mouse model and the results will be discussed.

Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (∼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (∼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.

Binding of fluorinated phenylalanine alpha-factor analogues to Ste2p: evidence for a cation-pi binding interaction between a peptide ligand and its cognate G protein-coupled receptor.

Ste2p, a G protein-coupled receptor (GPCR), binds alpha-factor, WHWLQLKPGQPMY, a tridecapeptide pheromone secreted by yeast cells. Upon alpha-factor binding, Ste2p undergoes conformational changes activating a signal transduction system through its associated heterotrimeric G protein leading to the arrest of cell growth in the G1 phase to prepare cells for mating. Previous studies have indicated that Tyr at position 13 of alpha-factor interacts with Arg58 on transmembrane one (TM1) of Ste2p. This observation prompted this investigation to determine whether a cation-pi type of interaction occurred between these residues. Tyrosine at position 13 of alpha-factor was systematically substituted with analogous amino acids with varying cation-pi binding energies using solid-phase peptide synthesis, and these analogues were modified by derivatization of their Lys(7) residue with the fluorescent group 7-nitrobenz-2-oxa-1,3-diazole (NBD) to serve as a useful probe for binding determination. Saturation binding of these peptides to Ste2p was assayed using whole yeast cells and a flow cytometer. In parallel the biological activities of the peptides were determined using a growth arrest assay. The data provide evidence for the presence of a cation-pi interaction between Arg58 of Ste2p and Tyr(13) of alpha-factor.

Discovery of pyrazolyl propionyl cyclohexenamide derivatives as full agonists for the high affinity niacin receptor GPR109A.

A series of pyrazolyl propionyl cyclohexenamides were discovered as full agonists for the high affinity niacin receptor GPR109A. The structure-activity relationship (SAR) studies were aimed to improve activity on GPR109A, reduce Cytochrome P450 2C8 (CYP2C8) and Cytochrome P450 2C9 (CYP2C9) inhibition, reduce serum shift and improve pharmacokinetic (PK) profiles.

Anthranilic acid replacements in a niacin receptor agonist.

Niacin is an effective drug for raising HDL cholesterol. However, niacin must be taken in large doses and significant side effects are often observed, including facial flushing, loss of glucose tolerance, and liver toxicity. An anthranilic acid was identified as an agonist of the niacin receptor. In order to improve efficacy and provide structural diversity, replacements for the anthranilic acid were investigated and several compounds with improved properties were identified.

HIV-1 peptide vaccine candidates: selecting constrained V3 peptides with highest affinity to antibody 447-52D.

The V3 region of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1) is a potential target for an anti-HIV-1 vaccine. Peptides corresponding to V3 form three variations of a beta-hairpin conformation when bound to anti-V3 HIV-1 neutralizing antibodies. The conformation of a V3(IIIB) peptide bound to the 0.5beta antibody, generated against an X4 gp120, has been postulated to represent the V3 conformation of X4 viruses while the conformations of a V3(MN) and a V3(CONSENSUS) peptide bound to the 447-52D human monoclonal antibody were postulated to represent the R5A and R5B V3 conformations of R5 viruses, respectively. To constrain the conformation of synthetic V3 peptides to these X4, R5A, and R5B conformations, we formed disulfide bonds between Cys residues whose location in a peptide template representing the entire V3(CONSENSUS) epitope recognized by the broadly neutralizing 447-52D antibody was changed systematically. In a previous study [Mor, A., et al. (2009) Biochemistry 48, 3288-3303] we showed that these constrained peptides adopted conformations resembling the three antibody-bound V3 conformations according to the location of the disulfide bonds. Here we show that these constrained peptides, with the exception of peptides in which the disulfide bond flanks the GPGR segment, retain high-affinity binding to the 447-52D antibody. Compared with peptides designed to mimic the X4 conformation, peptides designed to mimic either the R5A or R5B conformation had higher affinity to 447-52D. It is possible that constrained peptides which mimic the R5A and R5B conformations of the V3 and retain high-affinity binding to 447-52D are good candidates for eliciting a broad neutralizing antibody response similar to that of 447-52D.

Mimicking the structure of the V3 epitope bound to HIV-1 neutralizing antibodies.

The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or X4 phenotypes and use the CCR5 or CXCR4 chemokine coreceptor, respectively. Previous NMR studies revealed that both R5- and X4-V3 peptides bound to antibodies 0.5beta and 447-52D form beta-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3(JRFL) and V3(IIIB) peptides bound to 447-52D and to 0.5beta, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross-strand interactions suggestive of beta-hairpin-like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized beta-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity, and in one case, a structure close to that of V3(MN) peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3(IIIB) peptide bound to antibody 0.5beta was assumed.

Synthesis and biological evaluation of platensimycin analogs.

Platensimycin (1) displays antibacterial activity due to its inhibition of the elongation condensing enzyme (FabF), a novel mode of action that could potentially lead to a breakthrough in developing a new generation of antibiotics. The medicinal chemistry efforts were focused on the modification of the enone moiety of platensimycin and several analogs showed significant activity against FabF and possess antibacterial activity.